IL-23 comprises a p19 subunit and a p40 subunit that are disulfide-linked. IL-23 exerts its biological activities by binding to IL-23 receptor (IL-23R). IL-23R comprises an IL-23Rα subunit and an IL-12R81 subunit. When IL-23 binds to IL-23R, it leads to intracellular signaling including phosphorylation of STAT1, STAT3, STAT4 and STAT5. IL-23R is expressed on T-cells, NK cells, monocytes, and dendritic cells and its expression pattern corresponds with the ability of these cells to respond to IL-23. Mice lacking p19 exhibit a decreased pro-inflammatory response to experimental autoimmune encephalomyelitis, inflammatory bowel disease and collagen-induced arthritis. While IL-23 per se cannot induce the differentiation of naïve CD4 T-cells into Th-17 cells in vitro, the differentiation of Th17 cells in vivo may require IL-23. The observed protective effect in p19-deficient mice may relate to the lack of differentiation of Th17 cells.
IL-23Rα mRNA in human is 2.8 kb long and contains a total of 11 exons (NM_144701). The mature translated IL-23Rα protein is a type-I transmembrane protein (629 amino acids) and contains three (3) structural domains: (1) a signal peptide domain; (2) an extracellular region containing a fibronectin III-like domain; and (3) a 253 amino acid residue cytoplasmic domain with three (3) potential tyrosine phosphorylation sites.
While IL-23 is shown to bind to IL-23R and mediates Jak-STAT cell signaling, Parham et al. explicitly stated their inability to demonstrate human IL-23Rα-Ig and soluble human IL-23Rα-V5-His6 (composed of the entire extracellular domain—amino acids 1-353) as effective antagonists for human IL-23R. Daniel J. Cua et al. disclosed treatment methods for multiple sclerosis, neuropathic pain, and inflammatory bowel disorders using antibodies against IL-23 and its receptor. Contrary to Parham's statement, Cua proposes using a soluble receptor based on the extracellular region of a subunit of the IL-23 receptor (PCT/US2004/003126) as an antagonist. A recombinant human IL-23Rα Fc chimeric protein is commercially available (R&D Systems) and claimed to have the ability to inhibit IL-23 induced IL-17 secretion in a mouse splenocytes system. It remains unclear as to whether any of these proposed soluble IL-23R as may in fact exist in vivo as a naturally-occurring protein, let alone the possibility that such soluble IL-23R as may possess ability to block IL-23Rα mediated cell signaling. To this end, Daniel J. Cua et al. (PCT/US2004/003126) failed to provide any evidence that a soluble IL-23 receptor can indeed block IL-23 mediated cell signaling as well as inhibit Th17 producing cells.
Parham et al. disclosed the genomic organization of the IL-23R (composed of an IL-23α subunit and an IL-12W subunit). Recent evidence suggests that IL-23Rα gene may undergo extensive alternative mRNA splicing. There are at least twenty-four (24) potential gene transcripts for IL23Rα. From these IL-23Rα alternatively spliced mRNA sequences, there appears at least four (4) deduced putative translated proteins: (1) a short premature IL-23Rα extracellular peptide; (2) a possible soluble form of IL-23Rα lacking the transmembrane/intracellular domain; (3) a full-length IL23Rα with truncated extracellular region; and (4) a non-responsive membrane bound receptor isoform of IL-23Rα with deletion in intracellular signaling components.
Although many gene transcripts for IL-23Rα (i.e., IL-23Rα splice variants) are suggested, it is important to point out that their actual existence in vivo is unknown. There is little information regarding whether any of the deduced IL-23Rα translated products actually exist in vivo, let alone the function of these IL-23Rα protein variants, if any.
Duerr et al. first reported rs11209026 as an IL-23Rα coding variant (single nucleotide polymorphism, SNP) and associated the rs11209026 with protection against Crohn's disease (Science, Vol. 314 1 Dec. 2006). The mechanism by which SNP (rs11209026) functions is not known. Because rs11209026 is present on the exon 9 and results in amino acid change in the cytoplasmic domain of IL-23Rα, it is speculated that the SNP may affect IL-23Rα-mediated intracellular signaling.
To this end, Pidasheva et al. reported that the presence of rs11209026 interferes with the IL-23Rα intracellular signaling. Using the CD4+ T cells generated from healthy donors with wild-type and SNP rs11209026 haplotypes, this research group reported that donors with protective haplotype had significantly reduced STAT3 phosphorylation when compared to the wild-type counterparts. In contrast, de Paus et al. measured IL-23 signal transduction (i.e., STAT3) and IFN-γ and IL-10 production and found no detectable differences between the genetic variant (SNP rs11209026) and wild-type in the function of the IL-23Rα chain. (de Paus et al., 2008, Mol. Immunol., 45, 3889-95). These contradicting findings highlight the fact that the functional role of SNP rs 11209026 in IL-23Rα is far from clear.
Accordingly, there is continuing need for discovery of a therapeutic agent that inhibits IL-23 cellular signaling and antagonizes Th17 cell maturation. The present inventors have surprisingly discovered that the rs11209026 increases alternative splicing and leads to production of a naturally-occurring soluble form of IL-23Rα. The soluble form of IL-23Rα lacks the exon-9 of the IL-23Rα chain mRNA transcript (e.g., Δ9) and is found to be capable of blocking IL-23R signaling. The present inventors further discovered the use of anti-sense oligonucleotides to mimics rs11209026 to increase exon 9 skipping and increases production of Δ9. The present anti-sense oligonucleotides have the utility application to treat inflammatory bowel diseases such as Crohn's disease.